The immune system eliminates malignant cells by recognizing them as foreign and then clearing them from the body. To accomplish this, the immune system invokes both an antibody response and a cellular response. Both these responses require interaction among a number of different cells of the immune system (Abbas, Cellular and Molecular Immunology, 2000)
An immune reaction typically begins with a T lymphocyte (T cell) that has on its surface a T cell receptor (TCR) that binds to an antigen derived peptide associated with a class II major histo-compatability complex (MHC) molecule. The T cell also expresses on its surface various polypeptides, which are referred to as “ligands” because they bind to receptors on cells associated with an immune-mediated response, as described in more detail below. When the T cell receptor binds to a MHC-associated antigen, such as antigen derived from a malignant cell, it becomes activated and expresses a ligand on its surface. The ligand is only present on the cell surface for a short time, and once it has been removed from the surface of the cell, the T cell's ability to bind a receptor-bearing cell is lost. One such ligand is called tumor necrosis factor (TNFα).
TNFα, when expressed on the surface of an activated T cell, binds to receptors, such as TNF-receptor I (also known as “p55” or “CD120a”) and TNF-receptor II (also known as “p75” or “CD120b”), expressed on the surface of immune cells, non-immune cells, and malignant cells. Included among these immune cells are cells collectively referred to as “antigen presenting cells” (APC) because they express surface polypeptides that are able to bind and present antigen to the T cell. Examples of APC include dendritic cells and B cells. APC also have various receptor molecules on their surfaces that interact with other cells of the immune system. The interaction between ligands expressed by T cells and receptor molecules on APC and malignant cells causes a cytolytic reaction that destroys the malignant cells and clears them from the body.
TNFα is one member of a larger family of ligands, collectively referred to as the TNF superfamily (Gruss et al, Cytokines Mol Ther, 1:75-105, 1995 and Locksley et al, Cell, 104:487-501, 2001). Members of the TNF superfamily include Fas ligand (“FasL”), TNFα, LTα, lymphotoxin (TNFβ), CD154, TRAIL, CD70, CD30 ligand, 4-1BB ligand, APRIL, TWEAK, RANK ligand, LIGHT, AITR ligand, ectodysplasin, BLYS, VEGI, and OX40 ligand. TNF superfamily members share a conserved secondary structure comprising four domains: domain I, the intracellular domain; domain II, which spans the cell membrane and is known as the transmembrane domain; domain III, which consists of the extracellular amino acids closest to the cell membrane; and domain IV, the distal extracellular domain (Kipps et al., WO98/26061 published Jun. 18, 1998). Typically, at least a part of domain IV can be cleaved from the parent molecule. The cleaved fragment often exhibits the same biological activity of the intact ligand and is conventionally referred to as a “soluble form” of the TNF family member.
I. Biological Activity of TNFα.
There are two bioactive forms of TNFα. One form is membrane-integrated (mTNFα), also referred to as pro-TNFα. In addition, there is a soluble form (sTNFα) generated by proteolytic cleavage of mTNFα. TNF signals through two distinct receptors, CD120a and CD120b. In general, TNF signaling through CD120a induces cellular apoptosis due to the presence of a cytoplasmic death domain in CD120a. In contrast, CD120b, which lacks a death domain, generally induces cellular activation, such as proliferation and costimulatory molecule expression. These latter effects are highlighted in normal B cells in which TNFα, induced expression of important costimulatory molecules, including CD80 and CD54 (Ranheim and Kipps, Cell Immunol. 161:226, 1995).
A matrix metalloproteinase (mmp) called TACE (for TNF-alpha converting enzyme) has been shown to release the soluble form of TNFα (Black et al, Nature, 385:729-733, 1997 and Moss et al, Nature, 385:733-736, 1997). TACE has been found to release sTNFα by cleaving pro-TNFα between amino acid residues alanine76 and valine77. Moreover, this cleavage is dependent on an approximately 12 amino acid mmp recognition sequence spanning valine77 to proline88 (Decoster et al, J Biol Chem, 270:18473-18478, 1995 and Tang et al, Biochemistry, 35:8226-8233, 1996) since deletion of 9 to 12 amino acids of this mmp recognition site inhibited the cleavage of the parent TNFα molecule (Decoster et al, J Biol Chem, 270:18473-18478, 1995 and Perez et al, Cell, 63:251-258, 1990). However, deletion of this cleavage site does not necessarily completely abrogate sTNFα generation due to the existence of multiple cleavage sites in TNFα (Mueller et al, J Biol Chem, 274:38112-38118, 1999).
II. Drawbacks of Current TNFα Constructs in Treating Human Diseases
Since TNFα can induce apoptosis of CD120a expressing cells as well as enhance immune responses by cellular activation through CD120b, groups attempted to use TNFα as an anti-tumor compound. However, immune therapy of most cancers with recombinant soluble TNFα showed little clinical efficacy due to the failure to achieve high local concentrations of cytokine without systemic toxicity. Common side effects include fever, chills, anorexia, hypertension, liver abnormalities, and hematological changes (Spriggs et al, Ciba Found Symp, 131:206-227, 1987). Moreover, gene transfer of even wild-type (wt) TNF, expressed as the membrane-associated pro-TNFα, cannot achieve high local expression of TNF without systemic toxicity since it is metabolized rapidly into a soluble cytokine. Since the soluble form of TNFα is the common factor for the failure of TNFα as a therapeutic compound, we hypothesized that design of membrane-stabilized TNFα might allow local delivery of TNFα while mitigating the risk of systemic toxicity associated with soluble TNFα.
Given the disadvantages of current TNFα applications, there is clearly a need for a membrane-stabilized TNFα that maintains the receptor binding function of native TNFα but that is less susceptible to cleavage and is thereby less likely to generate the soluble form of TNFα. The present invention provides such a membrane-stabilized TNFα ligand.